Apoptosis, or programmed cell death, is a principal mechanism by which organisms eliminate unwanted cells. The deregulation of apoptosis, either excessive apoptosis or the failure to undergo it, has been implicated in a number of diseases such as cancer, acute inflammatory and autoimmune disorders, ischemic diseases and certain neurodegenerative disorders (see generally Science, 1998, 281, 1283–1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).
Caspases are a family of cysteine protease enzymes that are key mediators in the signaling pathways for apoptosis and cell disassembly (Thornberry, Chem. Biol., 1998, 5, R97–R103). These signaling pathways vary depending on cell type and stimulus, but all apoptosis pathways appear to converge at a common effector pathway leading to proteolysis of key proteins. Caspases are involved in both the effector phase of the signaling pathway and further upstream at its initiation. The upstream caspases involved in initiation events become activated and in turn activate other caspases that are involved in the later phases of apoptosis.
Caspase-1, the first identified caspase, is also known as interleukin converting enzyme or “ICE.” Caspase-1 converts precursor interleukin-1β (“pIL-1β”) to the pro-inflammatory active form by specific cleavage of pIL-1β between Asp-116 and Ala-117. Besides caspase-1 there are also eleven other known human caspases, all of which cleave specifically at aspartyl residues. They are also observed to have stringent requirements for at least four amino acid residues on the N-terminal side of the cleavage site.
The caspases have been classified into three groups depending on the amino acid sequence that is preferred or primarily recognized. The group of caspases, which includes caspases 1, 4, and 5, has been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site. Another group which includes caspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and 4 on the N-terminal side of the cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10, tolerate many amino acids in the primary recognition sequence, but seem to prefer residues with branched, aliphatic side chains such as valine and leucine at position 4.
The caspases have also been grouped according to their perceived function. The first subfamily consists of caspases-1 (ICE), 4, and 5. These caspases have been shown to be involved in pro-inflammatory cytokine processing and therefore play an important role in inflammation. Caspase-1, the most studied enzyme of this class, activates the IL-1β precursor by proteolytic cleavage. This enzyme therefore plays a key role in the inflammatory response. Caspase-1 is also involved in the processing of interferon gamma inducing factor (IGIF or IL-18) which stimulates the production of interferon gamma, a key immunoregulator that modulates antigen presentation, T-cell activation and cell adhesion.
The remaining caspases make up the second and third subfamilies. These enzymes are of central importance in the intracellular signaling pathways leading to apoptosis. One subfamily consists of the enzymes involved in initiating events in the apoptotic pathway, including transduction of signals from the plasma membrane. Members of this subfamily include caspases-2, 8, 9 and 10. The other subfamily, consisting of the effector capsases 3, 6 and 7, are involved in the final downstream cleavage events that result in the systematic breakdown and death of the cell by apoptosis. Caspases involved in the upstream signal transduction activate the downstream caspases, which then disable DNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton and finally fragment the cell.
A four amino acid sequence primarily recognized by the caspases has been determined for enzyme substrates. Talanian et al., J. Biol. Chem. 272, 9677–9682, (1997); Thornberry et al., J. Biol. Chem. 272, 17907–17911, (1997). Knowledge of the four amino acid sequence primarily recognized by the caspases has been used to design caspase inhibitors. Reversible tetrapeptide inhibitors have been prepared having the structure CH3CO—[P4]-[P3]-[P2]—CH(R)CH2CO2H where P2 to P4 represent an optimal amino acid recognition sequence and R is an aldehyde, nitrile or ketone capable of binding to the caspase cysteine sulfhydryl. Rano and Thornberry, Chem. Biol. 4, 149–155 (1997); Mjalli, et al., Bioorg. Med. Chem. Lett. 3, 2689–2692 (1993); Nicholson et al., Nature 376, 37–43 (1995). Irreversible inhibitors based on the analogous tetrapeptide recognition sequence have been prepared where R is an acyloxymethylketone—COCH2OCOR′. R′ is exemplified by an optionally substituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH2X where X is a leaving group such as F or Cl. Thornberry et al., Biochemistry 33, 3934 (1994); Dolle et al., J Med. Chem. 37, 563–564 (1994).
The utility of caspase inhibitors to treat a variety of mammalian disease states associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors. For example, in rodent models, caspase inhibitors have been shown to reduce infarct size and inhibit cardiomyocyte apoptosis after myocardial infarction, to reduce lesion volume and neurological deficit resulting from stroke, to reduce post-traumatic apoptosis and neurological deficit in traumatic brain injury, to be effective in treating fulminant liver destruction, and to improve survival after endotoxic shock. Yaoita et al., Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow and Metabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med., 184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585 (1999).
In general, the peptidic inhibitors described above are very potent against some of the caspase enzymes. However, this potency has not always been reflected in cellular models of apoptosis. In addition peptide inhibitors are typically characterized by undesirable pharmacological properties such as poor oral absorption, poor stability and rapid metabolism. Plattner and Norbeck, in Drug Discovery Technologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England, 1990).
There are reports of modified peptide inhibitors. WO 91/15577 and WO 93/05071 disclose peptide ICE inhibitors of the formula:Z-Q2-Asp-Q1wherein Z is an N-terminal protecting group; Q2 is 0 to 4 amino acids; and Q1 is an electronegative leaving group.
WO 99/18781 discloses dipeptide caspase inhibitors of the formula:
wherein R1 is an N-terminal protecting group; AA is a residue of a natural α-amino acid or β-amino acid; R2 is hydrogen or CH2R4 where R4 is an electronegative leaving group; and R3 is alkyl or hydrogen.
WO 99/47154 discloses dipeptide caspase inhibitors of the formula:
wherein R1 is an N-terminal protecting group; AA is a residue of a non-natural α-amino acid or β-amino acid; and R2 is optionally substituted alkyl or hydrogen.
WO 00/023421 discloses (substituted) acyl dipeptide apoptosis inhibitors having the formula:
where n is 0, 1, or 2; q is 1 or 2; A is a residue of certain natural or non-natural amino acid; B is a hydrogen atom, a deuterium atom, C1-10 straight chain or branched alkyl, cycloalkyl, phenyl, substituted phentyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH2)mcycloalkyl, (CH2)mphenyl, (CH2)m(substituted phenyl), (CH2)m(1- or 2-naphthyl), (CH2)mheteroaryl, halomethyl, CO2R13, CONR14R15, CH2ZR16, CH2OCOaryl, CH2OCO(substituted aryl), CH2OCO(heteroaryl), CH2OCO(substituted heteroaryl), or CH2OPO(R17)R18, where R13, R14, R15, R16, R17 and R18 are defined in the application; R2 is selected from a group containing hydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, (CH2)mNH2; R3 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; X is CH2, C═O, O, S, NH, C═ONH or CH2OCONH; and Z is an oxygen or a sulfur atom.
WO 97/24339 discloses inhibitors of interleukin-1β converter enzyme of the formula:
wherein R1 represents H, alkyl, alkoxy, a carbocycle, a heterocycle, and various other groups; AA1 and AA2 are single bonds or amino acids; and Y represents a group of formula:
wherein the Tet ring represents a tetrazole ring; and Z represents, inter alia, alkylene, alkenylene, O, S, SO, and SO2.
EP 618223 discloses ICE inhibitors of the formula:R-A1-A2-X-A3wherein R is H, a protecting group, or an optionally ring substituted PhCH2O; A1 is an α-hydroxy- or α-amino acid residue; A2 is an α-hydroxyacid residue or α-amino acid or A1 and A2 form together a pseudodipeptide or a dipeptide mimetic residue; X is a residue derived from Asp wherein A3 is CH2X1COY1, CH2OY2, CH2SY3 or CH2 (CO)mY6 wherein X1 is O or S, m is 0 or 1 and Y1, Y2, Y3 and Y6 are optionally substituted cyclic aliphatic or aryl groups.
WO 98/16502 discloses, inter alia, ICE inhibitors of the formula:
wherein R1 and R2 are as described in the application and the pyrrolidine ring is substituted by various groups.
While a number of caspase inhibitors have been reported, it is not clear whether they possess the appropriate pharmacological properties to be therapeutically useful. Therefore, there is a continued need for small molecule caspase inhibitors that are potent, stable, and penetrate membranes to provide effective inhibition of apoptosis in vivo. Such compounds would be extremely useful in treating the aforementioned diseases where caspase enzymes play a role.